Epidermal growth factor receptor (EGFR) gene expression is strictly regulated to maintain proper cell growth. We have identified nuclear proteins that act as activators or repressors of EGFR gene expression. We have cloned a cDNA for a repressor of EGFR gene expression termed GCF and developed and utilized an antiserum against the protein to show that it is a nuclear phosphoprotein that is more abundant in cells expressing low levels of EGFRs. We have cloned a second cDNA that has homology to the DNA binding region of GCF and named it GCF2. The GCF2 cDNA has an open reading frame of 2253 nucleotides. The deduced protein sequence contains 751 amino acids and has a calculated molecular weight of 83 kDa and a pI of 4.4. The cDNA hybridizes to an mRNA of 4.2 kilobases and is expressed at high levels in peripheral blood leukocytes and a Burkitt's lymphoma cell line (Raji). Expression is found in all human tissues examined but very low in testis and brain. An alternative size mRNA of 2.9 kb is found expressed at very high levels in skeletal muscle and is also present in heart tissue. A GCF2 His*Tag fusion protein expressed in bacteria cell and purified is able to bind EGFR promoter fragments and oligonucleotides containing GCF binding sites. TGF-beta1 increases the rate of EGFR transcription. We have localized the induction effect to a 60 base pair region between -919 to -860 in the EGFR promoter. Mutation analysis revealed that a CAGATG sequence in this region was required for binding interactions due to TGF-beta1 treatment of cells.